The present invention relates generally to a specific and quantitative test for hemoglobin including a method and apparatus for conducting such test and apparatus for collecting and preparing a sample for testing. More particularly, the invention relates to a test, and related method and apparatus, for quantitatively determining the level of hemoglobin in a biological material by converting the non-fluorescing heme portion of the hemoglobin to fluorescing porphyrin and assaying the fluorescence thereof. This test has particular applicability to a biological material such as feces or urine.
Various rapid screening tests for determining the presence of increased levels of hemoglobin in biological materials such as feces are currently available. These tests are used throughout the medical profession as the primary screening test for intestinal tumors. It is estimated that in excess of one million such tests are conducted each year in the United States for this purpose. Despite the fact that these tests do not yield quantitative data and that errors in test results are extremely costly, both personally and financially, and despite the fact that the tests currently available provide significantly high false positive and false negative results, their use is continued because there is no alternative.
The screening tests for hemoglobin in feces currently available do not involve converting the heme portion of hemoglobin to porphyrin and assaying its fluorescence; rather, currently available tests are indirect tests based on the peroxidase-like (pseudoperoxidase) activity of the hemoglobin. In these tests, colorless leuco dyes, in the presence of hemoglobin, become colored following addition of a suitable peroxide. Such tests, however, have several limitations. First, because of various factors including non-specificity and the fact that the reactivity is generally interferred with or affected by materials such as iron, ascorbic acid, or alterations in the hemoglobin molecule, significantly high false positive and false negative results are common. Secondly, interpretation of commercially available tests is often confusing because tests results are reported only as being "positive" or "negative". In addition to inherent differences in sensitivity of the different tests, the amount of feces included in test samples may easily vary by factors of 20 or more. These factors, as well as the above-noted non-specificities and differences in personal interpretation of color development, all contribute to limiting the usefulness of these tests. Because of these limitations, occult blood assay is among the few remaining non-quantitative tests in clinical and laboratory medicine.
Although no quantitative tests for hemoglobin in feces or urine involving the conversion of heme to protoporphyrin are currently available in the prior art, various studies have previously been done regarding this conversion. For example, in a study by G. R. Morrison in a paper entitled Fluorometric Microdetermination of Heme Protein, (Anal. Chem., 37:1124-1126, 1965) a method for measuring heme protein in animal tissues involving the conversion of heme to porphyrin through the use of oxalic acid with a subsequent assay for fluorescence was described. This method, however, was ineffective for quantitatively determining hemoglobin levels in excess of certain concentrations. Under the conditions described by Morrison, feces having elevated levels of hemoglobin would have to be diluted several thousand-fold. Such extreme dilution is not suitable for large-scale screening tests.
Accordingly, there is a need in the art for a quantitative test, including the method and apparatus for conducting such test and the apparatus for collecting and preparing the test sample, for determining the level of hemoglobin in biological materials such as feces or urine which eliminates or substantially reduces the incidence of false positives and false negatives and which is readily suitable for mass screening purposes.